The detection of 5-methyl cytosine in human DNA, usually at CpG dinucleotides, is important diagnostically because the methylation at such cytosines, particularly at gene control sequences, (e.g. promoter sequences) is frequently associated with the onset of cancer. This so-called epigenetic (since it is not in the usual sense heritable) modification of DNA is also important in development and frequently results in gene silencing. In cancer, the epigenetic change is aberrant and can result in the silencing of genes involved in the suppression of tumor formation, or alternatively the activation of genes involved in oncogenesis.
Current widely used methods to detect such DNA modification use treatment of DNA with the chemical bisulfite and have disadvantages with respect to performing a robust diagnostic assay. Among these are high complexity, the lengthy amount of time required, lack of reproducibility and significant loss of the DNA to be detected. In addition, the use of bisulfite is incompatible with the use of uracil-n-glycosylase in the control of carryover PCR product contamination. There is need for a method without these disadvantages.
At the same time there is a need for methods that detect such DNA modifications with high sensitivity and in the presence of high background levels of the same DNA sequence, unmodified. In a tumor, not all the cells contain DNA that is methylated at the sequence of interest—in fact, the majority of cells may not. Furthermore, in the case of early detection of cancer using either disseminated tumor cells or tumor DNA that can be found in the bloodstream, the vast majority of DNA is not methylated at the sequence of interest. At most, only a small percent of copies of that sequence may be methylated. The concentration of such sequences may be less than a single copy per milliliter of sample volume. The need for both high sensitivity and high specificity in detection is both clear and difficult to obtain by previous methods.